Trypanosomal TAC40 constitutes a novel subclass of mitochondrial β-barrel proteins specialized in mitochondrial genome inheritance

Abstract

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum-mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA-cytoskeleton linkage that is essential for mitochondrial DNA inheritance.

Keywords: organelle; parasitic protozoa.

Fig. 1.

TAC40 of procyclic cells localizes to the TAC. (A) Maximum-intensity projections of IF confocal microscopy images from whole procyclic T. brucei cells expressing C-terminally HA-tagged TAC40. Green, basal body region stained with the YL1/2 antibody; blue, DAPI-stained nuclear DNA and kDNA; red, HA-tagged TAC40. Three cell cycle stages containing one kDNA and one nucleus (1K1N, Top and Middle) or two kDNA and one nucleus (2K1N, Bottom) are shown. (Scale bar, 3 μm.) (B) The same as A but isolated flagella representing different cell cycle stages are shown (Left). Green, paraflagellar rod (PFR). (Scale bar, 3 μm.) (Right) three-dimensional volume reconstruction of the kDNA/TAC region from an isolated flagellum. (Scale bar, 1 μm.)

Fig. 2.

In vivo import-dependent accumulation of TAC40 depends on SAM50. (Left) Growth curve of uninduced (−Tet) and induced (+Tet) SAM50-RNAi cell line of procyclic T. brucei. (Right) Immunoblots of total cellular extracts from procyclic SAM50-RNAi cells that constitutively express HA-tagged TAC40 collected at the indicated times of induction. Immunoblots were probed for HA-tagged TAC40, the β-barrel protein VDAC, the non–β-barrel mitochondrial OM protein pATOM36, and LDH.

Fig. 3.

TAC40 is required for kDNA maintenance but not for kDNA replication. (A) Growth curve of uninduced (−Tet) and induced (+Tet) TAC40-RNAi cell lines of procyclic and bloodstream form T. brucei. (Insets) Northern blots demonstrating ablation of the TAC40 mRNA. Ethidium bromide-stained gel showing the rRNA region is used as a loading control. (B) Time course depicting the fraction of cells lacking kDNA during induction of TAC40-RNAi, as determined by microscopic examination of DAPI-stained cells. (C) IF images of uninduced (−Tet) and 2-d–induced (+Tet) TAC40-RNAi cells. Green, basal body region stained with the YL1/2 antibody; blue, DAPI-stained nuclear DNA and kDNA. For the uninduced population, five cell cycle stages consisting of cells containing one kDNA and one nucleus (1K1N), two kDNAs and one nucleus (2K1N), and two kDNAs and two nuclei (2K2N) are shown. The induced population in addition contains the abnormal cells containing no kDNA and one nucleus (0K1N) and cells lacking kDNA but having two nuclei (0K2N). (D) DAPI staining reveals large kDNA networks in the induced TAC40-RNAi cell line. (E) Quantification of the kDNA fluorescence intensity during induction of TAC40-RNAi in the kDNA-containing cell population. (F) Transmission electron micrographs showing the kDNA region of uninduced (−Tet) and 3-d–induced cells (+Tet).

Fig. 4.

TAC40 regulates neither mitochondrial morphology nor protein import. (A) Procyclic TAC40 and pATOM36 RNAi cell lines were tested for alterations of mitochondrial morphology. Maximum-intensity projections of IF confocal microscopy images of uninduced (−Tet) and induced (+Tet) cells stained with DAPI (blue) and with antibodies specific for the mitochondrial matrix protein HSP60 are shown. The position of the kDNA (K) is indicated. (Scale bars, 3 μm.) (B, Left) Duplicate immunoblots of digitonin-extracted crude mitochondrial fractions from the TAC40-RNAi cell line collected at the indicated times after induction of RNAi were separated on 4–13% blue native gradient gels (BN-PAGE) and probed with ATOM and cytochrome C1 (Cyt C1) antiserum detecting the ATOM and the bc1-complex, respectively. As a loading control a Coomassie-stained section of the same gels is shown. (Right) The same analysis as in Left, done for the SAM50 RNAi cell line.